Abstinent drug users remain at risk for relapse for long periods of time, well after their withdrawal symptoms subside. The reasons are poorly understood. Studies with laboratory animals offer a clue: responses to drug-related cues not only persist, but increase with abstinence duration. If this phenomenon, termed incubation of craving, also occurs in humans, it may contribute to prolonged relapse risk. We investigated this possibility in a translational study using a controlled experimental approach. Outpatient cigarette smokers were assigned to one of three different durations of verified, reinforced abstinence: 7, 14, or 35 days. Three groups were tested for cue reactivity at a single abstinence interval, and a fourth group was tested at all three intervals to investigate whether repeated exposure to cues would dampen responses to the cues over time. We hypothesized that smoking cue reactivity would increase with duration of abstinence after both single and repeated exposure to cues. The study was a collaborative effort conducted as a multisite trial at the University of Chicago (Dr. Harriet de Wit, PI) and the NIDA IRP. Smokers (healthy, non-treatment-seeking, adults who smoked at least 10 cigarettes daily, were randomized to four groups and paid to abstain for 7 (Group 1), 14 (Group 2), or 35 (Groups 3, 4) days. Abstinence was biochemically verified and withdrawal and mood questionnaires were collected in daily visits to the lab during the abstinence period. On the final abstinence day (Groups 1, 2, and 3), or on Days 7, 14 and 35 (Group 4), participants underwent a 2-hour cue session, during which they were exposed to smoking and neutral cues, in random order. Cues included visual, olfactory, and tactile stimuli. Eighty-six participants completed the study and were included in the analyses. As expected, craving increased more after the smoking than neutral cues. In both between- and within- groups analyses, cue-induced craving increased as a function of abstinence duration. Participants in Group 3 (35-day abstinence) reported significantly greater smoking-cue-elicited craving than did Group 1 participants (7-day abstinence). Participants in Group 4 (repeated cues) reported greater cue-elicited craving at 35 days than at 14 days (not shown). Time-dependent increases in conditioned craving occurred in the context of progressively decreasing baseline (non-provoked) craving and withdrawal symptoms. This is the first evidence of incubation-like changes in cue-induced craving in human drug users. These findings, which suggest that craving elicited by cues increase with abstinence even as daily background craving and nicotine withdrawal symptoms subside, have significant implications for treatment. Our findings indicate that clinicians and users should be aware that risk of relapse precipitated by cue-induced craving may increase with abstinence, even as withdrawal symptoms subside. Abstinent drug users may be at greater relapse risk when they return to the cue-laden environments after long absences from their normal environments such as after inpatient treatments. In other aspects of this project, we are focusing on stress, which has been shown to induce resumption of drug seeking in laboratory animals. Physical stress in the form of electric foot-shock has been consistently shown to lead to reinstatement of heroin and cocaine seeking in rats. This stress-induced reinstatement is blocked by the alpha-2 adrenergic receptor agonist clonidine. In one sense, the efficacy of alpha-2 agonists is specific to just one type of relapse: clonidine blocks stress-induced reinstatement, but not reinstatement induced by a drug-associated cue or a priming injection of drug. In another sense, however, the alpha-2 agonists may have a broad spectrum of efficacy: their blockade of stress-induced reinstatement has been demonstrated with heroin as well as cocaine. Thus, the alpha-2 agonists may act upon some final common pathway of stress-induced relapse, relevant to multiple drugs of abuse. We hypothesized that alpha-2 agonists would help prevent relapse in humans. We tested the effect of clonidine on stress- and cue-induced craving in human cocaine users in a laboratory study. Healthy, non-treatment-seeking cocaine users (n = 59) were randomly assigned to three groups receiving clonidine 0, 0.1, or 0.2 mg, orally, under double-blind conditions. In a single test session, each participants received study drug followed 3 hrs later by exposure to two pairs of standardized auditory-imagery scripts (neutral/stress and neutral/drug). Subjective measures of craving and saliva samples were collected at baseline and before and after script pairs. Drug and stress scripts significantly increased craving ratings. Responsivity to stress scripts was significantly attenuated in the 0.2 and 0.1 mg groups;responsivity to drug-cue scripts was significantly attenuated in the 0.2 mg group only. Clonidine also prevented a trend toward increases in salivary alpha-amylase associated with the drug script. Thus, clonidine may reduce cocaine craving and some physiological reactivity in drug abusers when they experience stress or (to a lesser degree) when they encounter cues that remind them of drug use. We are currently conducting a clinical trial of clonidine for the prevention of relapse to illicit opioid use in individuals in buprenorphine maintenance. We are using handheld electronic devices to improve outcome measurement. We have demonstrated the feasibility of this form of data collection in our population and are using it to determine whether clonidines relapse-prevention effect is specific to subtypes of relapse.